A paper copy of the Sequence Listing and a computer readable form (CRF) of the sequence listing on diskette, containing the file named xe2x80x9cseqlisting 16515-143.txtxe2x80x9d, which is 51.344 bytes in size (measured in MS-DOS), and which was recorded on Apr. 15, 2002, are herein incorporated by reference.
The present invention is directed to enzymes, methods to purify, and obtain such enzymes, amino acid and nucleic acid sequences related thereto, and methods of use for such compositions in genetic engineering applications.
Triacylglycerol (TAG) is thought to be the most important storage of energy for cells. Diacylglycerol acyl transferase is an enzyme which is believed to regulate TAG structure and direct TAG synthesis. The reaction catalyzed by DAGAT is at a critical branchpoint in glycerolipid biosynthesis. Enzymes at such branchpoints are considered prime candidates for sites of metabolic regulation. There are several enzymes which are common to the synthesis of diacylglycerol, TAG and membrane lipids, however, the DAGAT reaction is specific for oil synthesis.
In plants, TAG is the primary component of vegetable oil that is used by the seed as a stored form of energy to be used during seed germination. Higher plants appear to synthesize oils via a common metabolic pathway. Fatty acids are made in plastids from acetyl-CoA through a series of reactions catalyzed by enzymes known collectively as Fatty AcidSynthetase (FAS). The fatty acids produced in plastids are exported to the cytosolic compartment of the cell, and are esterified to coenzyme A. These acyl-CoAs are the substrates for glycerolipid synthesis in the endoplasmic reticulum (ER). Glycerolipid synthesis itself is a series of reactions leading first to phosphatidic acid (PA) and diacylglycerol (DAG). Either of these metabolic intermediates may be directed to membrane phospholipids such as phosphatidylglycerol (PG), phosphatidylethanolamine (PE) or phosphatidylcholine (PC), or they may be directed on to form neutral triacylglycerol (TAG).
Diacylglycerol (DAG) is synthesized from glycerol-3-phosphate and fatty acyl-CoAs in two steps catalyzed sequentially by glycerol-3-phosphate acyltransferase (G3PAT), and lysophosphatidic acid acyltransferase (LPAAT) to make PA, and then an additional hydrolytic step catalyzed by phosphatidic acid phosphatase (PAP) to make DAG. In most cells, DAG is used to make membrane phospholipids, the first step being the synthesis of PC catalyzed by CTP-phosphocholine cytidylyltransferase. In cells producing storage oils, DAG is acylated with a third fatty acid in a reaction catalyzed by diacylglycerol acyltransferase (DAGAT). Collectively, the reactions make up part of what is commonly referred to as the Kennedy Pathway.
The structure of the TAG, as far as positional specificity of fatty acids, is determined by the specificity of each of the three acyltransferases for the fatty acyl-CoA and the glycerol backbone substrates. Thus, for example, there is a tendency for the acyltransferases from many temperate zone species of seeds to allow either a saturated or an unsaturated fatty acid at the sn-1 or the sn-3 position, but only an unsaturated fatty acid at the sn-2. The absolute specificity for an unsaturated fatty acid at sn-2 is determined by the substrate preference of LPAAT enzyme. In some species such as cocoa, TAG compositions suggest that this tendency is carried further in that there is an apparent preference for acylation of the sn-3 position with a saturated fatty acid, if the sn-1 position is esterified to a saturated fatty acid. Thus, there is a higher percentage of structured TAG of the form SUS (where S=saturated fatty acid and U=unsaturated fatty acid), than would be expected from a random distribution based on the overall fatty acid composition with the sn-2 position fixed with an unsaturated fatty acid. This suggests that DAGAT also plays an important role in the regulation of TAG structure, if not also in the control of TAG synthesis.
Obtaining nucleic acid sequences capable of producing a phenotypic result in the incorporation of fatty acids into a glycerol backbone to produce an oil is subject to various obstacles including but not limited to the identification of metabolic factors of interest, choice and characterization of a protein source with useful kinetic properties, purification of the protein of interest to a level which will allow for its amino acid sequencing, utilizing amino acid sequence data to obtain a nucleic acid sequence capable of use as a probe to retrieve the desired DNA sequence, and the preparation of constructs, transformation and analysis of the resulting plants.
Thus, the identification of enzyme targets and useful tissue sources for nucleic acid sequences of such enzyme targets capable of modifying oil structure and quantity are needed. Ideally an enzyme target will be amenable to one or more applications alone or in combination with other nucleic acid sequences relating to increased/decreased oil production, TAG structure, the ratio of saturated to unsaturated fatty acids in the fatty acid pool, and/or to other novel oils compositions as a result of the modifications to the fatty acid pool.
For example, in some instances having an oilseed with a higher ratio of oil to seed meal would be useful to obtain a desired oil at lower cost. This would be typical of a high value oil product. Or such an oilseed might constitute a superior feed for animals. In some instances having an oilseed with a lower ratio of oil to seed meal would be useful to lower caloric content. In other uses, edible plant oils with a higher percentage of unsaturated fatty acids are desired for cardiovascular health reasons. And alternatively, temperate substitutes for high saturate tropical oils such as palm, coconut, or cocoa would also find uses in a variety of industrial and food applications.
In mammals, DAGAT plays an important role in the metabolism of cellular diacylglycerol and is important in processes involving triacylglycerol metabolism including intestinal fat absorption, lipoprotein assembly, adipose tissue formation and lactation. As such, identification and isolation of the DAGAT protein and of polynucleotide and polypeptide sequences is desired.
Several putative isolation procedures have been published for DAGAT. Polokoff and Bell (1980) reported solubilization and partial purification of DAGAT from rat liver microsomes. This preparation was insufficiently pure to identify a specific protein factor responsible for the activity. Kwanyuen and Wilson (1986, 1990) reported purification and characterization of the enzyme from soybean cotyledons. However, the molecular mass (1843 kDa) suggests that this preparation was not extensively solubilized and any DAGAT protein contained therein was part of a large aggregate of many proteins. Little et al (1993) reported solubilization of DAGAT from microspore-derived embryos from rapeseed, but as with Kwanyuen and Wilson, the molecular mass of the material that was associated with activity was so high, that complete solubilization is unlikely. Andersson et al (1994) reported solubilization and a 415-fold purification of DAGAT from rat liver using immunoaffinity chromatography. However, there is no evidence that the antibodies they used recognize DAGAT epitopes, nor that the protein that they purified is truly DAGAT. Indeed, as with Kwanyuen and Wilson, the DAGAT activity in their preparations exhibited a molecular mass typical of aggregated membrane proteins. Finally, Kamisaka et al (1993, 1994, 1996, 1997) report solubilization of DAGAT from Mortierella rammaniana and subsequent purification to homogeneity. They suggest that DAGAT solubilized from this fungal species has an apparent molecular mass of 53 kDa by SDS-PAGE. However, as shown in Example 4 below, fractions obtained using the protocol described by Kamisaka et al. did not provide abundant 53-kDa polypeptide which correlated with DAGAT activity.
The present invention is directed to diacyglycerol acyl transferase (DAGAT), and in particular to DAGAT polypeptides and polynucleotides. The polypeptides and polynucleotides of the present invention include those derived from plant, mammalian, including human, nematode and fungal sources.
In another aspect, the invention provides DAGAT proteins having molecular weights between approximately 36 kDa and 37 kDa, based on SDS-PAGE analysis, and particularly molecular weights of 36 kDa and 36.5 kDa. The preferred DAGAT proteins of the invention are obtainable from Mortierella ramanniana. 
In a further aspect the invention relates to oligonucleotides derived from the DAGAT proteins and oligonucleotides which include partial or complete DAGAT encoding sequences.
It is also an aspect of the present invention to provide recombinant DNA constructs which can be used for transcription or transcription and translation (expression) of DAGAT. In particular, constructs are provided which are capable of transcription or transcription and translation in plant and mammalian host cells. Particularly preferred constructs are those capable of transcription or transcription and translation in plant cells.
In another aspect of the present invention, methods are provided for production of DAGAT in a host cell or progeny thereof. In particular, host cells are transformed or transfected with a DNA construct which can be used for transcription or transcription and translation of DAGAT. The recombinant cells which contain DAGAT are also part of the present invention.
In a further aspect, the present invention relates to methods of using polynucleotide and polypeptide sequences to modify the ratios of oils to other constituents, as well as to modify the composition and/or structure of triglyceride molecules, particularly in seed oil of oilseed crops. Plant cells having such a modified triglyceride are also contemplated herein.
The modified plants, seeds and oils obtained by the expression of the plant DAGAT proteins are also considered part of the invention.
In a further aspect, the invention relates to methods for using such polypeptides and polynucleotides in mammals are provided. Such methods include treating or ameliorating diseases associated with DAGAT activity, including diseases associated with altered cellular diacylglycerol concentration or protein kinase C activity, including, but not limited to cancer; diabetes; cardiopulmonary diseases including, but not limited to heart failure, and atherosclerosis; adipocytosis; leukemia and skin carcinoma; fibroblastoma; metabolic disorders; obesity; diseases associated with abnormal lipid metabolism; diseases associated with abnormal fat absorption, lipoprotein secretion and adipogenesis. In addition, methods are provided for altering the levels of DAGAT activity.
In another aspect of the present invention, methods for identifying agonists and antagonists/inhibitors of DAGAT, and treating conditions associated with DAGAT activity or altering levels of DAGAT activity with such agonists or antagonists are provided.
It is also an aspect of the present invention to provide diagnostic assays for detecting alterations in the level of DAGAT activity and for diagnosing conditions associated with DAGAT activity.